Revisiting the effect of PCR replication and sequencing depth on biodiversity metrics in environmental DNA metabarcoding.

Environmental DNA (eDNA) metabarcoding is an increasingly popular tool for measuring and cataloguing biodiversity. Because the environments and substrates in which DNA is preserved differ considerably, eDNA research often requires bespoke approaches to generating eDNA data. Here, we explore how two experimental choices in eDNA study design-the number of PCR replicates and the depth of sequencing of PCR replicates-influence the composition and consistency of taxa recovered from eDNA extracts. We perform 24 PCR replicates from each of six soil samples using two of the most common metabarcodes for Fungi and Viridiplantae (ITS1 and ITS2), and sequence each replicate to an average depth of ~84,000 reads. We find that PCR replicates are broadly consistent in composition and relative abundance of dominant taxa, but that low abundance taxa are often unique to one or a few PCR replicates. Taxa observed in one out of 24 PCR replicates make up 21-29% of the total taxa detected. We also observe that sequencing depth or rarefaction influences alpha diversity and beta diversity estimates. Read sampling depth influences local contribution to beta diversity, placement in ordinations, and beta dispersion in ordinations. Our results suggest that, because common taxa drive some alpha diversity estimates, few PCR replicates and low read sampling depths may be sufficient for many biological applications of eDNA metabarcoding. However, because rare taxa are recovered stochastically, eDNA metabarcoding may never fully recover the true amplifiable alpha diversity in an eDNA extract. Rare taxa drive PCR replicate outliers of alpha and beta diversity and lead to dispersion differences at different read sampling depths. We conclude that researchers should consider the complexity and unevenness of a community when choosing analytical approaches, read sampling depths, and filtering thresholds to arrive at stable estimates.